KR20240055159A - Treatment for gingivitis - Google Patents

Abstract

The present invention relates to compositions and their use for oral care. In particular, the compositions and methods are intended to maintain oral health and/or treat various oral diseases such as gingivitis. The present invention reduces pathogenic oral bacteria in the oral cavity of an individual; increasing commensal oral bacteria in an individual's oral region; Reduces the rate of pathogenic oral bacteria in an individual's oral region; Suppresses oral bacterial imbalance; Reduces gingival inflammation in individuals in need of reducing gingival inflammation; treating gingivitis in an individual in need thereof; It relates to a method of treating chronic gingivitis in a subject in need thereof and the use of stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP) in the manufacture of a medicament therefor.

Description

TREATMENT FOR GINGIVITIS {TREATMENT FOR GINGIVITIS}

Cross-reference to related applications

This application claims priority from Australian Provisional Application No. 2017900893, the entire contents of which are incorporated by reference in their entirety.

The present invention relates to compositions and their use for oral care. In particular, the compositions and methods are intended to maintain oral health and/or treat various oral diseases such as gingivitis.

Cross-reference to related applications

This application claims priority from Australian Provisional Application No. 2017900893, the entire contents of which are incorporated by reference in their entirety.

The present invention relates to compositions and their use for oral care. In particular, the compositions and methods are intended to maintain oral health and/or treat various oral diseases such as gingivitis.

In one aspect, the present invention provides a method of reducing pathogenic oral bacteria in the oral cavity of a subject, comprising administering stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP) to the oral cavity of the subject. Reducing pathogenic bacteria in the oral cavity by administering the drug to the oral cavity.

In another aspect, the present invention also provides a method of increasing commensal oral bacteria in an oral region of an individual, the method comprising using stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP). and increasing commensal oral bacteria in the oral cavity by administering to the oral cavity of the subject.

In another aspect, the present invention also provides a method of reducing the rate of pathogenic oral bacteria in the oral cavity of an individual, the method comprising the use of stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP). ) to the oral cavity of the subject, thereby reducing the proportion of pathogenic oral bacteria in the oral cavity.

In any of the aspects of the invention described herein, the pathogenic oral bacteria may be any one or more associated with gingival inflammation, gingivitis, chronic gingivitis, periodontitis, or periodontal disease. Typically, pathogenic oral bacteria are acidogenic and/or acid tolerant and/or inflammatory. Preferably, the bacteria are inflammatory. Bacteria that produce lipopolysaccharide (LPS) and release LPS into tissues as LPS are highly inflammatory.

Preferably, the bacteria are Streptococcus mutans, Actinomyces naeslundii, Veillonella parvula, Lactobacillus casei, Porphyromonas Porphyromonas gingivalis , Tannerella forsythia, Treponema denticola, Leptotrichia wadei , Leptothrichia shahii , Leptotri Any one or more selected from Leptotrichia buccalis and Lautropia mirabilis.

In any aspect of the invention described herein, the commensal oral bacteria can be any one or more species that express arginine deiminase and/or nitrate reductase. Typically, the bacteria are Corynebacterium durum , Rothia dentocariosa, Streptococcus mitis, Streptococcus sanguinis and Fusobacterium nuclei. Any one or more of Fusobacterium nucleatum .

In one aspect, the present invention provides a method for inhibiting oral bacterial imbalance, comprising administering stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP) to the oral cavity of a subject. It includes steps to suppress bacterial imbalance.

In another aspect, the present invention provides a method of reducing gingival inflammation in a subject in need thereof, comprising the use of stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP). ) comprising reducing gingival inflammation by administering to the oral cavity of the subject. Preferably, the method further comprises an initial step of identifying an individual with gingival inflammation.

In another aspect, the present invention provides a method of treating gingivitis in a subject in need thereof, comprising the use of stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP). and treating gingivitis by administering it to the oral cavity of the subject. Preferably, the method further comprises an initial step of identifying an individual with gingivitis.

In another aspect, the present invention provides a method of treating chronic gingivitis in a subject in need thereof, comprising: stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP) ) includes treating chronic gingivitis by administering to the oral cavity of the subject. Preferably, the method further comprises an initial step of identifying an individual with chronic gingivitis.

In another aspect, the present invention provides a method of treating periodontitis in an individual in need thereof, comprising: stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP). It includes treating periodontitis by administering it to the oral cavity of the subject. Preferably, the method further comprises an initial step of identifying an individual with periodontitis.

In any aspect of the invention, the method further comprises performing a dental procedure prior to administering the stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP) to the oral cavity of the individual. . Examples of dental procedures include debridement, scaling, root planning, or any other procedure to remove subgingival or supragingival bacteria.

In another aspect, the invention provides the use of stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP) in the manufacture of a medicament:

- Medications to reduce pathogenic oral bacteria in the oral region of an individual;

- Medications to increase commensal oral bacteria in the oral region of an individual;

- Medications to reduce the proportion of pathogenic oral bacteria in the oral region of an individual

- Medicines to suppress oral bacterial imbalance;

- Medications for reducing gingival inflammation in individuals in need thereof;

- Medicines for treating gingivitis in individuals in need of such treatment; or

- A medicine for the treatment of chronic gingivitis in individuals in need of such treatment.

In another aspect, the present invention provides stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP) for use in:

- Reducing pathogenic oral bacteria in the oral area of an individual;

- increasing commensal oral bacteria in the oral region of an individual;

- Reducing the proportion of pathogenic oral bacteria in the oral region of an individual

- Suppressing oral bacterial imbalance;

*- Reducing gingival inflammation in individuals in need thereof;

- Treating gingivitis in individuals in need of gingivitis treatment; or

- To treat chronic gingivitis in individuals in need of treatment.

In another aspect, the present invention provides a method of reducing demineralization of tooth enamel in a subject, comprising administering stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP) to the subject. Reducing demineralization of tooth enamel in the subject by administering to the oral cavity. Preferably, the stabilized ACP complex is a tin-bound phosphopeptide (PP) stabilized amorphous calcium phosphate (ACP) complex, and the stabilized ACFP complex is a tin-bound phosphopeptide (PP) stabilized amorphous calcium phosphate (ACP) complex. It is an amorphous calcium fluoride phosphate (ACFP) complex. Preferably, the reduction in demineralization is a reduction in the rate of demineralization.

Preferably, the stabilized amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) are phosphopeptide stabilized. Preferably, the phosphopeptide (as defined below) is a casein phosphopeptide.

In any of the methods or uses of the invention, the stabilized ACP or ACFP complex may be administered to the subject for 5 to 60 minutes, 10 to 45 minutes, 10 to 30 minutes, or 20 minutes. Additionally, the stabilized ACP or ACFP complex can be administered 4, 5 or 6 times per day, or per 24 hour period. Preferably, the stabilized ACP or ACFP complex is administered for a period of 1 to 2 weeks.

In some aspects, the compositions include toothpastes, mouthwashes, mouthwashes, mouth sprays, varnishes, dental cements, troches, chewing gum, dental products, including cream toothpastes, tooth powders, and liquid toothpastes. It can be prepared and used in a variety of forms applicable to the mouth, such as pastes, creams for gingival massage, tablets for gargling, dairy products and other foods including yogurt. Preferably, the composition is chewing gum. Preferably, the chewing gum contains at least about 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg or 60 mg of stabilized ACP or ACFP complex. A chewing gum may contain about 18.8 or 56.4 mg of stabilized ACP or ACFP complex.

In some aspects, the calcium ion content of the stabilized ACP or ACFP complex is greater than about 30 moles per mole of PP. Preferably, the calcium ion content ranges from about 30 to 100 moles of calcium per mole of PP. More preferably, the calcium ion content ranges from about 30 to about 50 moles of calcium per mole of PP.

In some aspects, the stabilized ACP complex is a tin-bound phosphopeptide (PP) stabilized amorphous calcium phosphate (ACP) complex, and the stabilized ACFP complex is a tin-bound phosphopeptide (PP) complex. It is a stabilized amorphous calcium fluoride phosphate (ACFP) complex.

*In some aspects, the ACP and/or ACFP complex is in the form of a casein phosphopeptide stabilized ACP and/or ACFP complex.

Preferably, the phase of the ACP is predominantly (i.e. >50%) a basic phase, wherein the ACP primarily comprises the species Ca ²⁺ , PO4 ^3- and OH ^- . The basic phase of ACP may have the general formula [Ca ₃ (PO ₄ ) ₂ ] _x [Ca ₂ (PO ₄ )(OH)], where x ≥ 1. Preferably x = 1 to 5. More preferably, x = 1, i.e. the two components of the formula are present in equal proportions. Accordingly, in one embodiment, the basic phase of ACP has the formula Ca ₃ (PO ₄ ) ₂ Ca ₂ (PO ₄ )(OH).

Preferably, the phase of ACFP is predominantly (i.e. >50%) a basic phase, wherein the ACFP mainly comprises the species Ca ²⁺ , PO ₄ ^3- and F ^- . The basic phase of ACFP may have the general formula [Ca ₃ (PO ₄ ) ₂ ] _x [Ca ₂ (PO ₄ )F] _y , where x ≥ 1 when y = 1 or y ≥ 1 when x = 1 am. Preferably, y = 1 and x = 1 to 3. More preferably, y = 1 and x = 1, i.e. both components of the formula are present in equal proportions. Accordingly, in one embodiment, the basic phase of ACFP has the formula Ca ₃ (PO ₄ ) ₂ Ca ₂ (PO ₄ )F.

In one embodiment, the ACP complex consists essentially of phosphopeptide, calcium, phosphate and hydroxide ions, and water. Preferably, the complex further comprises stannous ions.

In one embodiment, the ACFP complex consists essentially of phosphopeptide, calcium, phosphate, fluoride and hydroxide ions, and water. Preferably, the complex further comprises stannous ions.

*The present invention also relates to a kit for use in the methods or uses of the present invention, the kit comprising:

(a) a composition described herein, or

(b) A stabilized ACP or ACFP complex described herein.

As used herein, except when the context otherwise requires, the term “comprise” and variations of the term such as “comprising,” “comprising,” and “included” mean additional It is not intended to exclude additives, ingredients, integers or steps.

Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example, and with reference to the accompanying drawings.

Figure 1 . Effect of CPP-ACP on plaque index in a randomized controlled clinical trial.
Figure 2 . Impact of CPP-ACP on gingival index in a randomized controlled clinical trial.
Figure 3. Average species composition of six ascites biofilms cultured with human enamel substratum in a constant depth film fermentor pulsed with 1% sucrose four times per day. All six species, namely, Actinomyces naeslundii (An), Fusobacterium nucleatum (Fn), Lactobacillus casei (Lc), Streptococcus mutans (Sm), and Streptococcus sanguinis ( Ss) and Veillonella parbula (Vp) were detected at each time point. Streptococcus mutans remained relatively constant over 19 days and was the most abundant species. Actinomyces naeslundii and Lactobacillus casei increased in abundance over time, while Streptococcus sanguinis decreased. This is consistent with ascites biofilms becoming more acidic over time. The three bars above each species representation represent relative abundance at 6 days (black), 12 days (gray), and 19 days (stripes) after inoculation.
Figure 4. Enamel subsurface demineralization in ascites biofilm caries model. Figure 4a. Integrated mineral loss (vol% min. μm) of the enamel matrix over a 19-day period in a multimicrobial caries model. Data are representative of four biological replicates of control (untreated) and are presented as mean ± SD. Figure 4b. Representative transverse micrographs of the enamel substrate showing subsurface demineralization at days 6, 12, and 19. Figure 4c. Representative electron micrograph of ascitic biofilm from day 12 showing tight association of bacterial biofilm and supragingival plaque-like structures.
Figure 5. Effect of twice daily addition of SnF ₂ , 2% CPP-ACP and 2% CPP-ACP-SnF ₂ on ascites biofilm bacterial species composition in CDFF at day 19. Figure 5a. Bacterial species composition as a percentage of total bacteria in the biofilm (data and statistical analysis presented in Table 7). Figure 5b. Changes in abundance of each species after treatment compared to control. Fusobacterium nucleatum is not shown due to its extremely high relative increase in ascites biofilms treated with 2% CPP-ACP-SnF ₂ at 4,981%. SnF ₂ treatment had no effect on Fusobacterium nucleatum abundance (-2%), whereas CPP-ACP treatment resulted in a 355% increase at day 19. Striped bars = control, black = SnF ₂ , gray = 2% CPP-ACP, and white = 2% CPP-ACP-SnF ₂ . Figure 5c. Representative 3D-rendered CLSM image of ascites biofilm treated with CPP-ACP-SnF ₂ at day 19. Bacterial cells were stained with four species-specific FISH probes (purple - Fusobacterium nucleatum; blue - Actinomyces naeslundii; red - Streptococcus mutans; green - Streptococcus sanguinis).

It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example, and with reference to the accompanying drawings.

Reference will now be made in detail to specific embodiments of the invention. Although the invention will be described in conjunction with embodiments, it will be understood that the invention is not intended to be limited to these embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications and equivalents that may fall within the scope of the invention as defined by the claims.

Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The invention is not limited in any way to the methods and materials described.

All patents and publications referenced herein are incorporated by reference in their entirety.

For the purposes of interpreting this specification, terms used in the singular also include the plural and vice versa.

As used herein, except when the context otherwise requires, the term “comprise” and variations of the term such as “comprising,” “comprising,” and “included” mean additional It is not intended to exclude additives, ingredients, integers or steps. As used herein, “comprise” and “include” may be used interchangeably, except where the context requires otherwise.

The present invention is based on the unexpected discovery that stabilized amorphous forms of calcium phosphate, such as CPP-ACP, CPP-ACFP or stannous-linked ACP or ACFP, can reduce gingival inflammation and treat various gingival diseases such as gingivitis. . Stabilized amorphous calcium phosphate forms have already been shown to remineralize dental lesions by delivering calcium and phosphate to form crystalline hydroxyapatite or fluoroapatite in dental lesions, but these stabilized amorphous forms of calcium phosphate are surprisingly effective in oral and It has now been shown to affect both beneficial and pathogenic oral bacteria present in tissues within the oral cavity (i.e. gingiva). More surprisingly, the stabilized amorphous form of calcium phosphate increases the relative abundance of beneficial oral bacteria while decreasing the relative abundance of pathogenic oral bacteria. Without being bound by any theory or mechanism of action, it appears that this differential effect on oral bacteria results in a reduction in gingival inflammation.

Any of the methods of the invention may include treatment of an oral region of the subject, wherein the oral region is distal-buccal, mid-buccal, mesial-buccal, mesial-palatal, mid-palatal, and distal-palatal and distal. -Any one or more areas around the teeth, including lingual, medio-lingual and mesio-lingual. The treatment may be applied directly to the gingiva and not to other areas of the mouth. Treatment can be multiple oral sites. Alternatively, the entire oral cavity can be treated. Additionally, the efficacy of a treatment can be determined by analysis of one or more oral regions or the entire oral cavity. For example, a decrease in pathogenic bacteria or an increase in commensal bacteria can be determined by analysis of one or more oral regions described herein or by analysis of the entire oral cavity.

In any aspect of the invention, the subject is an individual in need of treatment or prevention. Specifically, in any aspect of the invention, the method or use further comprises identifying an individual in need of treatment or prevention.

Individuals in need of treatment to reduce pathogenic oral bacteria or increase commensal oral bacteria may be affected by modern lifestyles (e.g., excessive dietary sugar intake, smoking, poor oral hygiene) or other factors (e.g., genetics). An individual may have or is experiencing a disruption of the oral microbiome due to a predisposition.

In any aspect of the invention, the individual may not have any dental lesions. For example, an individual may be identified as having gingival inflammation or gingivitis but no detectable tooth surface or subsurface lesions.

The term “treat” or “treatment” refers to therapeutic treatment aimed at slowing (reducing) undesirable physiological changes or disorders. For the purposes of this invention, a favorable or desired clinical outcome, whether detectable or undetectable, includes relief of symptoms, reduction in disease severity, stabilization (i.e., no worsening) of the disease, or progression of the disease. These include, but are not limited to, delaying or slowing, improving or alleviating disease conditions, and remission (whether partial or complete). Treatment may not necessarily result in the complete absence of detectable symptoms of the disease, but it may reduce or minimize the complications and side effects of the disease. The success of treatment, etc. can be monitored by physical examination of the individual, cytopathology, serological DNA, mRNA detection techniques, or any other technique described herein.

The terms “prevent” and “prophylaxis” generally refer to prophylactic or prophylactic measures to prevent or prevent an individual not having gingival inflammation or any other disease described herein from developing its complications. . Individuals in need of prevention include individuals with bacterial imbalance.

The phrase “pharmaceutically acceptable” indicates that a substance or composition must be chemically and/or toxicologically compatible with the other ingredients that make up the formulation and/or the subject being treated therewith.

The methods of the present invention are applicable to individuals exhibiting subclinical or clinical symptoms of a disease or disorder of the oral tissue described herein.

Dysbiosis in an individual means that the individual has an abnormal total amount or relative abundance of oral microbial pathogens in the context of a randomly selected cohort of individuals. For example, an individual may have an increased amount or proportion of one or more pathogenic bacteria associated with gingival inflammation, gingivitis, chronic gingivitis, periodontitis, or periodontal disease. Typically, pathogenic bacteria are acidogenic and/or acid tolerant and/or inflammatory. Preferably, the bacteria are from Streptococcus mutans, Actinomyces naeslundii, Veillonella parvula, Lactobacillus casei, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. One or more randomly selected items.

Symptoms of gingival inflammation may appear in an individual's oral tissues in more than one oral region. Cellular features of inflammation that may be present include increased migration of plasma and white blood cells from the blood into damaged tissue. Gingival inflammation, including rubor (redness), color (increased heat), tumor (swelling), dolor (pain), and functio laesa (loss of function) Clinical signs may also be present. Chronic inflammation may be characterized by infiltration of white blood cells (monocytes, macrophages, lymphocytes, plasma cells). Tissue and bone loss may be observed. Examples of inflammation include gingivitis.

Reducing gingival inflammation may result in a decrease in the incidence and/or severity of gingival inflammation. This may be determined by a reduction in the incidence or severity of any of the clinical, cellular, or biochemical characteristics described herein, such as those outlined immediately above.

In a further embodiment, the individual may exhibit chronic inflammation of oral tissues. In one example, an individual may exhibit gingivitis (e.g., chronic gingivitis), resorption of alveolar bone and eventual tooth loss due to progressive loss of the tooth's collagen attachment to the alveolar bone. Other lesions of the mucosa or associated oral tissues are possible.

In some aspects, an individual in need may be an individual identified as having mild, moderate, or severe gingival inflammation. Because chronic gingivitis can lead to more severe periodontitis, the present invention also provides treatment for mild, moderate and severe periodontitis as determined by the CDC-AAP methodology described in Eke et al J Dent Res 91:914-920 (2012). It may be applicable to entities having . The invention is also applicable to individuals with gingival inflammation confirmed using the Modified Gingival Index (Lobene et al 1986 Clin Prev Dent. Jan-Feb; 8(1):3-6). do. This index is ) and Silness gingival index and allows greater discrimination of mild and moderate gingivitis. An individual may be determined to have gingival inflammation on a scale of 0 to 4 for gingival tissue involving one or more sites (e.g., mesial and distal buccal, lingual). Preferably, the individual in need thereof has a modified gingival index score of 1, 2, 3 or 4. Accordingly, in any aspect of the invention, an individual is provided having a degree of gingival inflammation described herein. In any aspect of the invention, the method or use further comprises determining whether the individual has the degree of gingival inflammation described herein.

Identification of individuals in need of a reduction in pathogenic oral bacteria, an increase in commensal oral bacteria, or an individual with an oral dysbiosis can be determined by the amount or relative proportions of bacteria in a sample obtained from oral fluid collected from the oral cavity. In particular, the oral fluid may be saliva, gingival crevicular fluid, or blood. Oral fluid, such as saliva, is recognized as a combination of secretions from multiple sources such as the parotid, submandibular, sublingual, accessory glands, gingival mucosa, and buccal mucosa, and the term oral fluid refers to secretions from each of these sources individually. or in combination. Saliva may be stimulated or, in preferred embodiments, may be non-stimulated. Stimulation of saliva in a subject can occur by having the subject chew a piece of sugar-free gum, paraffin film, or tart candy. Unstimulated saliva means that the subject will spit into the collection container without stimulation of saliva flow.

Saliva specimens for testing can be collected according to a variety of methods known in the art, for example, stimulated or unstimulated saliva by an individual spitting into a collection container or by extracting the saliva using a cotton swab or syringe. It can be sampled by doing Other methods for obtaining unstimulated saliva are known in the art (Nazaresh and Christiansen, J. Dent. Res. 61: 1158-1162 (1982)). Methods and devices for collecting saliva have also been described (see also U.S. Pat. No. 5,910,122).

It is contemplated that the method of the present invention may also be practiced by analyzing stimulated saliva.

Additionally, the method of the present invention is not limited to performing saliva analysis immediately after sample collection. In certain implementations, saliva analysis according to the methods of the present invention may be performed on archived saliva samples. Saliva samples for testing can be preserved using methods and devices known in the art (see, eg, U.S. Pat. No. 5,968,746).

The method of the present invention is also contemplated for use in performing saliva analysis on saliva samples that have been treated to reduce viscosity.

The viscous nature of saliva, due to the nature of mucopolysaccharides, makes testing of these fluids difficult. To prepare saliva for any laboratory testing procedure, the saliva must be sufficiently fluid (i.e., reduced in viscosity) and free of debris. Techniques used to remove debris include centrifugation and filtration. The viscosity of saliva can also be reduced by mixing the saliva sample with a cationic quaternary ammonium reagent (see U.S. Pat. No. 5,112,758).

In another embodiment, a sample from an individual may be taken from a crypt on the dorsum of the tongue.

Samples from an individual may be taken from a specific periodontal site. The periodontal region is the area within the oral cavity. Preferably, the periodontal region is around the teeth including distal-buccal, mid-buccal, mesial-buccal, mesial-palatal, mid-palatal and distal-palatal and distal-lingual, mid-lingual and mesial-lingual. is the area of Samples may be taken from periodontal areas showing clinical signs of inflammation.

In another embodiment, the sample from the individual may be a sample of tissue. The tissue or portion thereof may be from the oral cavity. In certain embodiments, the tissue is gingiva. Gingival tissue is derived from various areas around the teeth, including distal-buccal, mid-buccal, mesial-buccal, mesial-palatal, mid-palatal and distal-palatal and distal-lingual, mid-lingual and mesial-lingual. It may be of. Tissue may be obtained by normal biopsy or may be obtained from an extracted tooth.

In another embodiment, the sample from the individual may be dental plaque. Plaque may be subgingival or supragingival. Subgingival plaque can be sampled using a sterile curette or paper point. Supragingival plaque can be isolated using standard techniques known in the art. Subgingival plaque is around the teeth, including the distal-buccal, mid-buccal, mesial-buccal, mesial-palatal, mid-palatal and distal-palatal and distal-lingual, mid-lingual, and mesial-lingual periodontal areas. It can be collected from various parts. Subgingival plaque samples may be obtained during a normal dental examination provided by a qualified dentist or periodontal specialist. Plaque samples can be analyzed as is or processed to extract proteins, peptides, or fragments thereof of interest using extraction buffer. The extraction buffer contains a pH buffer (e.g. phosphate, HEPES, etc.), a salt to maintain ionic strength (e.g. NaCl) and a protein solubilizer (e.g. detergent (SDS, Triton X100, etc.)), and a reducing agent. (e.g., dithiothreitol, cysteine HCl) and/or chaotropic agents (e.g., urea, guanidinium chloride, lithium perchlorate).

Dysbiosis is a comparison of the amount or relative proportions of pathogenic and/or commensal oral bacteria in a sample from an individual and compares it to a set of previously defined parameters or arbitrary parameters of gingival inflammation from an individual that does not have the attributes of an individual with dysbiosis. It can be determined by comparing clinical, cellular, or biochemical characteristics. It is considered that individuals with healthy oral cavity may contain low levels of pathogenic bacteria present. This low or normal level of pathogenic bacterial colonization does not indicate dysbiosis. When using such a control and comparing it to a test sample, a determination of whether an individual has a bacterial imbalance can be determined by (1) an elevated level of one or more pathogenic bacteria described herein in a sample taken from the individual compared to the control sample, or ( 2) an increased proportion of one or more pathogenic bacteria in a sample taken from an individual compared to the total level of bacteria in a control sample, or (3) one or more different bacterial species in a sample taken from an individual compared to a control sample. Contains an increased proportion of pathogenic bacteria.

The stabilized-ACP or ACFP complex referred to herein includes the stabilized-ACP or ACFP complex described in International Patent No. PCT/AU2005/001781, the content of which is incorporated by reference.

In a preferred embodiment, the phosphopeptide stabilized amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) complex has tightly bound and loosely bound calcium, wherein the bound calcium in the complex has a pH of 7.0. Of the ACP or ACFP complexes formed, there is less than tightly bound calcium. Optionally, ACP or ACFP exists primarily in basic form.

Stabilized-ACP or ACFP complexes referred to herein include stabilized-ACP or ACFP complexes formed at a pH of less than 7.0. Preferably the complex is formed at a pH ranging from about 5.0 to less than 7.0. More preferably the complex is formed at a pH ranging from about 5.0 to about 6.0. In a preferred embodiment, the complex is formed at a pH of about 5.5. Preferably, the ACP or ACFP in the complex exists primarily in basic form.

Stabilized-ACP can be produced by a method comprising the following steps:

(i) obtaining a solution comprising one or more phosphopeptides;

(ii) mixing the solutions containing calcium ions, phosphate ions, and hydroxide ions while maintaining the pH at about 7.0 or less.

Stabilized ACFP can be produced by a method comprising the following steps:

(i) obtaining a solution comprising one or more phosphopeptides;

(ii) mixing solutions containing calcium ions, phosphate ions, hydroxide ions, and fluoride ions while maintaining the pH at about 7.0 or less.

Phosphopeptide stabilized amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) complexes may also include those obtainable or obtained by a method comprising the following steps, wherein the ACP in the complex is tightly bound. There is less tightly bound calcium in the complex than the tightly bound calcium in the ACP or ACFP complex formed at a pH of 7.0, and ACP or ACFP exists primarily in the basic form:

a) a first solution comprising calcium ions, a second solution comprising phosphate ions, and optionally a third solution comprising fluoride ions, comprising a phosphopeptide and mixing in a solution containing a solvent; and

b) maintaining the pH of the solution below about 5.0 and 7.0 by addition of hydroxide ions during mixing.

“Tightly” and “loosely” bound calcium and phosphate in ACP or ACFP can be determined using analytical ultrafiltration. Briefly, the solution of the mixed phosphopeptide, calcium, phosphate and optionally fluoride is first filtered through a 0.1 micrometer filter to remove free calcium and phosphate not associated with the complex while maintaining the pH below about 7.0. can do. This free calcium and phosphate is present in the filtrate, which is discarded. Any free calcium or phosphate that is not associated with the complex in any way is not bioavailable, i.e., is delivered to the teeth by phosphopeptides. The retentate from the 0.1 micrometer filtration can be further analyzed by centrifugation through a 3000 mw cutoff filter for 15 minutes at 1,000 g. The resulting filtrate contains calcium and phosphate loosely bound or bound to the complex. At this centrifugal force, calcium and phosphate that are not tightly bound to the complex are liberated and migrate into the filtrate. The tightly bound Ca and Pi in the complex are retained in the retentate. The amount of tightly bound Ca and Pi in the retentate can then be determined by subtracting the amounts of Ca and Pi in the filtrate from the total amount of Ca and Pi in the retentate of the 0.1 micrometer filtration.

Stabilized-ACP or ACFP complexes referred to herein include the stabilized-ACP or ACFP complexes described in International Patent No. PCT/AU2006/000885, the content of which is incorporated by reference.

“Superloaded” phosphopeptide or phosphoprotein (PP) stabilized-amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) complexes. Complexes can be formed at any pH (eg, 3 to 10). Preferably the phosphopeptide comprises the sequence -A-B-C-, where A is a phosphoamino acid, preferably phosphoserine, B is any amino acid comprising a phosphoamino acid, and C is glutamic acid, aspartic acid or It is a phosphoamino acid. The phosphoamino acid may be phosphoserine. PP is superloaded with calcium and phosphate ions. Calcium ions may range from 30 to 1000 moles of Ca per mole of PP, or from 30 to 100 or 30 to 50 moles of Ca per mole of PP. In another embodiment, the moles of Ca per mole of PP is at least 25, 30, 35, 40, 45 or 50.

The phosphopeptide or phosphoprotein (PP) stabilized amorphous calcium phosphate or amorphous calcium fluoride phosphate complex may have a calcium ion content greater than about 30 moles of calcium per mole of PP. In a preferred embodiment, the calcium ion content ranges from about 30 to 100 moles of calcium per mole of PP. More preferably, the calcium ion content ranges from about 30 to about 50 moles of calcium per mole of PP.

Phosphopeptide or phosphoprotein (PP) stabilized-amorphous calcium phosphate (ACP) or amorphous calcium fluoride phosphate (ACFP) complexes can be produced by a method comprising the following steps:

(i) obtaining solutions comprising calcium, inorganic phosphate and (optionally) fluoride; and

(ii) mixing (i) with a solution containing PP-ACP.

In a preferred embodiment, PP is casein phosphopeptide (CPP).

The PP stabilized ACP and/or ACFP complex may further comprise at least an equal amount by weight of calcium phosphate. Preferably the calcium phosphate is CaHPO ₄ . Preferably, calcium phosphate (eg CaHPO ₄ ) is dry blended with the PP stabilized ACP and/or ACFP complex. In a preferred embodiment, the ratio of PP-ACP and/or PP-ACFP complex:calcium phosphate is about 1:1 to 50, more preferably about 1:1 to 25, more preferably about 1:5 to 15. . In one embodiment, the ratio of PP-ACP and/or PP-ACFP complex:calcium phosphate is about 1:10.

Comprising a phosphopeptide or phosphoprotein (PP) stabilized amorphous calcium phosphate (ACP) and/or amorphous calcium fluoride phosphate (ACFP) complex having a calcium ion content greater than about 30 moles of calcium per mole of calcium when used orally. Oral care formulations can be produced by a method comprising the following steps:

(i) obtaining a powder comprising PP-ACP and/or PP-ACFP complex;

(ii) dry blending with an effective amount of calcium phosphate; and

(iii) formulating the dry blended PP-ACP and/or mixture of PP-ACFP and calcium phosphate into an oral care formulation.

Preferably, the form of calcium phosphate for dry blending is any soluble calcium phosphate including, but not limited to, CaHPO ₄ , Ca ₂ HPO ₄ and calcium lactate.

The compositions described herein may further include free fluoride ions. The fluoride ion may be from any suitable source. The source of fluoride ions may include free fluoride ions or fluoride salts. Examples of sources of fluoride ions include, but are not limited to: sodium fluoride, sodium monofluorophosphate, stannous fluoride, sodium silicofluoride, and amine fluoride. They may be provided as solutions (typically aqueous solutions) or suspensions.

Fluoride ions are preferably present in the composition in an amount greater than 1 ppm. More preferably, the amount is greater than 3 ppm. In another embodiment, the amount is preferably greater than 10 ppm. In typical embodiments described below, the amounts may be hundreds or thousands of ppm. Typically the fluoride content is measured as ppm in oral compositions in a manner commonly used in the art. When the fluoride is provided from a source comprising stabilized ACP, ppm represents the concentration of fluoride in that source, typically a solution or suspension of bioavailable fluoride.

Annotation-bound ACP or ACFP complexes referred to herein include any described in International Patent No. PCT/AU2014/050447, the entire content of which is incorporated by reference.

Compositions described herein for use in the methods of use of the present invention may comprise stannous ACP or ACFP complexes. The composition may include 2% CPP-ACP and 290 ppm fluoride with 220 ppm fluoride as stannous fluoride and 70 ppm as sodium fluoride.

“Phosphopeptide” in the context of the present description means an amino acid sequence in which one or more amino acids are phosphorylated. Preferably, the phosphopeptide comprises one or more of the amino acid sequence -A-B-C-, where A is a phosphoamino residue, B is any amino acyl residue including a phosphoamino residue, and C is glutamyl , aspartyl or phosphoamino residues. Any of the phosphoamino residues may independently be a phosphoseryl residue. B is preferably a residue whose side chain is neither relatively large nor hydrophobic. This may be Gly, Ala, Val, Met, Leu, Ile, Ser, Thr, Cys, Asp, Glu, Asn, Gln or Lys.

In another embodiment, at least two of the phosphoamino acids in the sequence are preferably contiguous. Preferably the phosphopeptide comprises the sequence A-B-C-D-E, where A, B, C, D and E are independently phosphoserine, phosphothreonine, phosphotyrosine, phosphohistidine, glutamic acid or aspartic acid, and At least two, preferably three, of B, C, D and E are phosphoamino acids. In a preferred embodiment, the phosphoamino acid residue is phosphoserine, most preferably three contiguous phosphoserine residues. It is also preferred that D and E are independently glutamic acid or aspartic acid.

In one embodiment, ACP or ACFP is stabilized by casein phosphopeptide (CPP), present in the form of intact casein or fragments of casein, and the complex formed preferably has the formula [CPP(ACP) ₈ ] _n or [( CPP)(ACFP) ₈ ] _n , where n is 1 or more, for example 6. The complex formed may be a colloidal complex, where the core particles aggregate to form large (eg, 100 nm) colloidal particles that are suspended in water. Accordingly, PP may be a casein protein or a phosphopeptide.

PP may be from any source; It may exist in the context of larger polypeptides, including full-length casein polypeptides, by tryptic digestion or other enzymatic or chemical digestion of casein, or other phosphoamino acid-rich proteins, such as phosphithin, or by chemical or recombinant synthesis. provided that it comprises the sequence -ABC- or ABCDE as described above. The sequences flanking this core sequence can be any sequence. However, the flanking sequences α _s1 (59-79), β (1-25), α _s2 (46-70) and α _s2 (1-21) are preferred. The flanking sequences may optionally be modified by deletion, addition, or conservative substitution of one or more residues. The amino acid composition and sequence of the flanking regions are not critical.

Examples of conservative substitutions are illustrated in Table A below.

[Table A]

The flanking sequences may also include non-natural amino acid residues. Commonly encountered amino acids that are not coded for by the genetic code include:

2-amino adipic acid (Aad) for Glu and Asp;

2-aminopimelic acid (Apm) for Glu and Asp;

2-aminobutyric acid (Abu) for Met, Leu, and other aliphatic amino acids;

2-aminoheptanoic acid (Ahe) for Met, Leu and other aliphatic amino acids;

2-aminoisobutyric acid (Aib) for Gly;

Val, and cyclohexylalanine (Cha) for Leu and Ile;

Homoarginine (Har) for Arg and Lys;

2, 3-diaminopropionic acid (Dpr) for Lys, Arg and His;

N-ethylglycine (EtGly) for Gly, Pro, and Ala;

N-ethylasparigine (EtAsn) for Asn, and Gln;

Hydroxylysine (Hyl) for Lys;

Allohydroxylysine (AHyl) for Lys;

3-(and 4) hydroxyprolines (3Hyp, 4Hyp) for Pro, Ser, and Thr;

Aloysoleucine (Alle) for Ile, Leu, and Val;

ρ-amidinophenylalanine for Ala;

N-methylglycine (MeGly, sarcosine) for Gly, Pro, and Ala;

N-methylisoleucine (MeIle) for Ile;

norvaline (Nva) for Met and other aliphatic amino acids;

norleucine (Nle) for Met and other aliphatic amino acids;

Ornithine (Orn) for Lys, Arg and His;

citrulline (Cit) and methionine sulfoxide (MSO) for Thr, Asn, and Gln;

For Phe, N-methylphenylalanine (MePhe), trimethylphenylalanine, halo (F, Cl, Br and I) phenylalanine, trifluorylphenylalanine.

In one embodiment, PP is divided into α _s1 (59-79) [1], β (1-25) [2], α _s2 (46-70) [3] and α _s2 (1-21) [4]. It is one or more phosphopeptides selected from the group consisting of:

[1] Gln ⁵⁹ -Met-Glu-Ala-Glu-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ile-Val-Pro-Asn-Ser( P)-Val-Glu-Gln-Lys ⁷⁹ α _s1 (59-79)

[2] Arg ¹ -Glu-Leu-Glu-Glu-Leu-Asn-Val-Pro-Gly-Glu-Ile-Val-Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser (P)-Glu-Glu-Ser-Ile-Thr-Arg ²⁵ β(1-25)

[3] Asn ⁴⁶ -Ala-Asn-Glu-Glu-Glu-Tyr-Ser-Ile-Gly-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser(P)-Ala- Glu-Val-Ala-Thr-Glu-Glu-Val-Lys ⁷⁰ α _s2 (46-70)

[4] Lys ¹ -Asn-Thr-Met-Glu-His-Val-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser-Ile-Ile-Ser(P)-Gln- Glu-Thr-Tyr-Lys ²¹ α _s2 (1-21)

In another embodiment of the invention, the stabilized ACP and/or stabilized ACFP complex is incorporated into an oral composition, such as a cream toothpaste, mouthwash, or oral formulation to help prevent and/or treat gingivitis or periodontitis. . The oral composition comprises a layer comprising stabilized ACP and/or ACFP in an amount sufficient to form a layer on the tooth surface, preferably said layer having a calcium:phosphate ratio equivalent to that of normal apatite, for example, The ratio is approximately 2:1. The layer may contain calcium in an amount of about 20% by weight. The stabilized ACP and/or ACFP complex is present in an amount of 0.01 to 50%, preferably 1.0 to 50%, preferably 1.0 to 30%, preferably 1.0 to 20%, preferably 1.0 to 10%, based on the weight of the composition. %, preferably 2 to 10%. In a particularly preferred embodiment, the oral compositions of the present invention contain about 2% of stabilized ACP or ACFP complex or a mixture of both. Oral compositions of the present invention containing the above agents include toothpastes including cream toothpastes, tooth powders and liquid toothpastes, mouthwashes, mouthwashes, oral sprays, varnishes, dental cements, troches, chewing gum, dental pastes, It can be manufactured and used in various forms applicable to the mouth, such as gingival massage cream, gargle tablets, dairy products, and other foods. The oral composition according to the present invention may further comprise additional well-known ingredients depending on the type and form of the particular oral composition. Certain compositions of the invention, such as cream toothpastes, toothpastes and liquid toothpastes, mouthwashes, mouthwashes and mouth sprays, have relatively low viscosity and have a positive therapeutic or prophylactic effect without significant residence time in the oral cavity.

In certain preferred forms of the invention, the oral composition may be substantially liquid in nature, such as a mouthwash, mouthwash or oral spray. In such formulations, the vehicle is typically a water-alcohol mixture, preferably comprising a humectant, as described below. Typically, the water:alcohol weight ratio ranges from about 1:1 to about 20:1. Typically the total amount of water-alcohol mixture in these types of formulations ranges from about 70 to about 99.9% by weight of the formulation. The alcohol is typically ethanol or isopropanol. Ethanol is preferred.

In another preferred form of the invention, the composition may be substantially solid or paste-like in character, such as tooth powder, dental tablets or cream-type toothpaste (dental cream) or gel-type toothpaste. The vehicle of such solid or paste oral preparations generally contains dentally acceptable polishing materials. Examples of polishing materials include water-insoluble sodium metaphosphate, potassium metaphosphate, tricalcium phosphate, dihydrate calcium phosphate, dicalcium phosphate anhydrous, calcium pyrophosphate, magnesium orthophosphate, trimagnesium phosphate, calcium carbonate, hydrated alumina, calcined alumina, There are aluminum silicate, zirconium silicate, silica, bentonite, and mixtures thereof. Other suitable polishing materials include particulate thermosets such as melamine-, phenolic, and urea-formaldehyde, and crosslinked polyepoxides and polyesters. The preferred polishing material has a particle size of about 5 microns or less, an average particle size of about 1.1 microns or less, and a surface area of about 50,000 cm ² /g. It includes crystalline silica, silica gel or colloidal silica, and complex amorphous alkali metal aluminosilicates.

When visually clear gels are used, polishing agents of colloidal silica, such as Syloid 72 and Syloid 74 sold under the trade names Syloid, or Santocel 100 sold under the trade names Santosel; Alkali metal aluminosilicate complexes are particularly useful because they have a refractive index close to that of the gelling agent-liquid (comprising water and/or humectant) systems commonly used in toothpastes.

Many of the so-called “water-insoluble” polishing materials are anionic in nature, and they also contain small amounts of soluble material. Accordingly, insoluble sodium metaphosphate can be prepared in any suitable manner, for example in Thorpe's Dictionary of Applied Chemistry, Volume 9, 4th Edition, pp. 510-511]. The insoluble forms of sodium metaphosphate known as Madrell's salt and Kurrol's salt are further examples of suitable materials. These metaphosphate salts exhibit only minimal solubility in water and are therefore commonly referred to as insoluble metaphosphate (IMP). There is a small amount of soluble phosphate material present therein as an impurity, generally less than 4% by weight. In the case of insoluble metaphosphate, the amount of soluble phosphate material believed to contain soluble sodium trimetaphosphate can be reduced or eliminated, if desired, by washing with water. Insoluble alkali metal metaphosphates are typically used in powder form with particle sizes such that less than 1% of the material is larger than 37 microns.

Typically, the polishing material is present in a solid or paste-like composition in a weight concentration of about 10% to about 99%. Preferably, it is present in an amount of about 10% to about 75% in the cream toothpaste and in an amount of about 70% to about 99% in the tooth powder. In cream toothpastes, when the polishing material is siliceous in nature, it is generally present in amounts of about 10 to 30% by weight. Other polishing materials are typically present in amounts of about 30 to 75 weight percent.

In cream toothpastes, the liquid vehicle may include water and humectant, typically in amounts ranging from about 10% to about 80% by weight of the formulation. Glycerin, propylene glycol, sorbitol and polypropylene glycol are examples of suitable humectants/carriers. Also advantageous are liquid mixtures of water, glycerin and sorbitol. In transparent gels where refractive index is an important consideration, about 2.5 to 30% (w/w) water, 0 to about 70% (w/w) glycerin, and about 20 to 80% (w/w) sorbitol are preferred. It is used extensively.

Creamy toothpastes, creams and gels typically contain natural or synthetic thickening or gelling agents in a proportion of about 0.1 to about 10, preferably about 0.5 to about 5% (w/w). Suitable thickeners include synthetic hectorites, synthetic colloidal magnesium alkali metal silicate complex clays (e.g. Laponite sold by Laporte Industries Limited (e.g. CP, SP 2002, D) is available as). Laponite D is approximately 58.00% SiO ₂ , 25.40% MgO, 3.05% Na ₂ O, 0.98% Li ₂ O, and some water and trace metals by weight. Its true specific gravity is 2.53, and it has an apparent bulk density of 1.0 g/ml at 8% moisture.

Other suitable thickeners are Irish moss, iota carrageenan, gum tragacanth, starch, polyvinylpyrrolidone, hydroxyethylpropylcellulose, hydroxybutyl methyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose ( available, for example, as Natrosol), sodium carboxymethyl cellulose, and colloidal silicas such as micronized siloids (e.g., 244). Solubilizing agents, such as humectant polyols, such as propylene glycol, dipropylene glycol and hexylene glycol, cellosolves, such as methyl cellosolve and ethyl cellosolve, vegetable oils and waxes containing about 12 or more carbons in the straight chain, such as olive oil, Castor oil and petrolatum and esters such as amyl acetate, ethyl acetate and benzyl benzoate may also be included.

It will be appreciated that, as is customary, oral preparations are generally sold or otherwise distributed in appropriately labeled packages. Accordingly, a jar of mouthwash will in fact have a label describing it as a mouthwash or mouthwash and with instructions for its use; Creamy toothpastes, creams or gels generally come in extruded tubes, typically aluminum, lined lead or plastic, or other squeeze tubes, with a label describing them as creamy toothpastes, gels or dental creams in nature. ), may be present in a pump or pressurized dispenser (for metering out the contents).

Organic surface active agents can be used in the compositions of the invention to achieve increased prophylactic action and to help achieve thorough and complete dispersion of the active agents throughout the oral cavity, making the compositions of the invention more cosmetically acceptable. . The organic surface active material is preferably anionic, nonionic or amphoteric in nature and preferably does not interact with the active agent. As a surface active agent, it is preferred to use a detergent material that imparts detergent and foaming properties to the composition. Suitable examples of anionic surfactants include water-soluble salts of higher fatty acid monoglyceride monosulfates, such as sodium salts of monosulfated monoglycerides of hydrogenated coconut oil fatty acids, higher alkyl sulfates such as sodium lauryl sulphate. pates, alkyl aryl sulfonates such as sodium dodecyl benzene sulfonate, higher alkylsulfo-acetates, higher fatty acid esters of 1,2-dihydroxy propane sulfonate, and higher substantially saturated lower aliphatic amino carboxylic acid compounds. Aliphatic acyl amides, such as those having 12 to 16 carbons in the fatty acid, alkyl or acyl radical. Examples of the last-mentioned amides are N-lauroyl sarcosine, and the sodium, potassium and ethanolamine salts of N-lauroyl, N-myristoyl, or N-palmitoyl sarcosine (which include soap or similar higher-grade salts). must be substantially free of fatty acid materials). The use of these sarconite compounds in the oral compositions of the present invention is particularly advantageous because, in addition to the fact that these materials exert some reduction in the solubility of tooth enamel in acid solutions, acid formation in the oral cavity due to the decomposition of carbohydrates This is because it shows a clear effect over a long period of time in suppressing . Examples of water-soluble nonionic surfactants suitable for use include condensation products of ethylene oxide and various reactive hydrogen-containing compounds with long hydrophobic chains (e.g., aliphatic chains of about 12 to 20 carbon atoms) that are reactive therewith. The condensation product (“ethoxamer”) is a hydrophilic polyoxyethylene moiety, such as poly(ethylene oxide), and a fatty acid, fatty alcohol, fatty amide, or polyhydric alcohol (e.g., sorbitan). monostearate) and polypropylene oxide (e.g., Pluronic materials).

Typically the surface active agent is present in an amount of about 0.1 to 5% by weight. It is noteworthy that surface active agents can assist in dissolving the active agent of the invention thereby reducing the amount of solubilizing humectant required.

Various other materials such as whitening agents, preservatives, silicones, chlorophyll compounds and/or ammoniated materials such as urea, diammonium phosphate, and mixtures thereof may be included in the oral formulations of the present invention. Such adjuvants, if present, are included in the formulation in amounts that do not substantially adversely affect the desired properties and characteristics.

Any suitable flavoring or sweetening agent may also be used. Examples of suitable flavoring ingredients include flavoring oils such as spearmint, peppermint, wintergreen, sassafras, clove, sage, eucalyptus, marjoram, cinnamon, lemon. , and oil of orange, and methyl salicylate. Suitable sweeteners include sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate, perillartine, AMP (aspartyl phenyl alanine, methyl ester), saccharin, and the like. Suitably, flavoring and sweetening agents, individually or together, may comprise about 0.1% to 5% or more of the formulation.

The compositions of the present invention may also be incorporated into lozenges or chewing gums or other products, for example, by stirring into a warm gum base or forming a gum base (examples of which include jelutong, rubber latex, vinylite resin, etc.). ), preferably together with conventional plasticizers or softeners, sugar or other sweeteners or such as glucose, sorbitol, etc. The compositions of the present invention may be dual phase compositions, where each phase allows release of the components over a different period of time.

An alternative composition may be to provide stabilized ACP or ACFP and a stannous compound which then forms stannous-linked stabilized ACP or ACFP in situ, such as in the oral cavity. An exemplary composition may be a chewing gum containing stabilized ACP or ACFP within the pellets and a stannous compound within the central chew.

In a further aspect, the invention provides a composition comprising a pharmaceutical composition comprising a composition comprising a stabilized ACP or ACFP complex as described above in combination with a compound capable of increasing or maintaining the pH of the solution and a pharmaceutically acceptable carrier. provides. Such compositions may be selected from the group consisting of dental, anti-caries and therapeutic compositions. Dental or therapeutic compositions may be in the form of gels, liquids, solids, powders, creams or lozenges. Therapeutic compositions may also be in the form of tablets or capsules. In one embodiment, the stabilized ACP or ACFP complex is substantially the only active ingredient of such compositions. For example, a cream formulation containing: water; glycerol; CPP-ACP/SnF ₂ ; D-sorbitol; silicon dioxide; Sodium Carboxymethylcellulose (CMC-Na); propylene glycol; titanium dioxide; xylitol; phosphoric acid; guar gum; sodium saccharin; ethyl p-hydroxybenzoate; magnesium oxide; Butyl p-hydroxybenzoate and propyl p-hydroxybenzoate.

The present invention further provides that the above-described formulation is provided with instructions for use thereof for treating or preventing any one or more of dental caries or cavities, dental erosion and fluorosis, dentinal hypersensitivity, plaque, gingivitis or periodontitis. Includes.

In another embodiment, the compositions of the invention described herein do not include phosphate buffering agents and/or calcium chelating agents. For example, any toothpaste described herein may not include phosphate buffering agents and/or calcium chelating agents.

In certain embodiments of the invention, compositions are provided, wherein the composition does not include phosphate buffering agents and/or calcium chelating agents.

In another embodiment, the compositions of the invention described herein do not include a viscosity modifier, or from 0.5 to 50% of a viscosity modifier.

In another embodiment, the compositions of the invention described herein do not include sodium carboxymethylcellulose, or 0.01 to 10% sodium carboxymethylcellulose with a degree of esterification of 0.7 to 1.0.

In one embodiment, the active ingredient of the composition consists essentially of stabilized ACP or ACFP complex.

Although this specification specifically refers to applications in humans, it will be clearly understood that the invention is also useful for veterinary purposes. Accordingly, in all respects the present invention relates to livestock, such as cattle, sheep, horses and poultry; companion animals such as cats and dogs; and useful for zoo animals.

One example of a mineralizing composition includes (in order of decreasing proportions):

water

glycerol

CPP-ACP/SnF ₂

D-Sorbitol

silicon dioxide

Sodium Carboxymethylcellulose (CMC-Na)

propylene glycol

titanium dioxide

xylitol

phosphoric acid

guar gum

sodium saccharin

Ethyl p-hydroxybenzoate

Magnesium Oxide

Butyl p-hydroxybenzoate

Propyl p-hydroxybenzoate

The present invention also provides kits comprising stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP), said kits being suitable for use in the methods described above.

Kits may include:

- a container containing a composition comprising stabilized amorphous calcium phosphate (ACP) and/or stabilized amorphous calcium fluoride phosphate (ACFP);

-Label or package insert with instructions for use.

In certain embodiments, the kit may contain one or more additional active elements or ingredients for treatment of a disease or condition.

A kit may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. Containers can be formed from various materials such as glass or plastic. The container contains a therapeutic composition effective for treating a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). . The label or package insert indicates that the therapeutic composition is used to treat the condition of choice. In one embodiment, the label or package insert includes instructions for use and indicates that the therapeutic composition can be used to treat a given disease or condition.

The kit includes (a) a therapeutic composition; and (b) a second container containing a second active ingredient or ingredient. The kit in this embodiment of the invention may further include a package insert indicating that the compositions and other active ingredients can be used to treat disorders or diseases described herein or to prevent complications due to bacterial imbalance. Alternatively or additionally, the kit may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. . It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The invention will now be further illustrated with reference to the following non-limiting examples.

Example

Example 1

Clinical Study: Prebiotic Effects of CPP-ACP on Supragingival Plaque and Oral Health

Research Objectives

primary goal

On the microbial composition of supragingival plaque formed on maxillary molars, chewing sugar-free gum containing 56.4 mg CPP-ACP and chewing sugar-free gum containing 18.8 mg CPP-ACP, together with normal oral hygiene practices for 14 days each. To compare the effects of chewing gum and not chewing gum.

secondary goal

A. For the development of supragingival plaque, chewing sugar-free gum containing 56.4 mg CPP-ACP, chewing sugar-free gum containing 18.8 mg CPP-ACP, and gum, along with normal oral hygiene practices for 14 days each. This is to compare the effects of not chewing.

B. Chewing sugar-free gum containing 56.4 mg CPP-ACP, chewing sugar-free gum containing 18.8 mg CPP-ACP, and chewing gum, each combined with normal oral hygiene practices for 14 days, for the development of gingivitis. To compare the effects of not chewing.

Human ethics approval

Human ethics approval was obtained from the University of Melbourne's Human Research Ethics Committee before starting the study. All participants were required to provide written informed consent before starting the study.

research plans

study design

This investigator-blinded, randomized controlled clinical trial examined chewing sugar-free gum containing 56.4 mg CPP-ACP over 14 days on the development and composition of supragingival plaque as well as the development of gingivitis in the presence of normal oral hygiene practices. A three-treatment, three-period crossover design was used to evaluate the effects of chewing sugar-free gum containing 18.8 mg CPP-ACP and not chewing gum. Participants were randomly assigned to one of three treatments, with each treatment lasting 14 days. The three treatments were as follows:

A. Chew sugar-free gum containing 56.4 mg CPP-ACP for 20 minutes, 6 times per day for 14 consecutive days;

B. Chewing sugar-free gum containing 18.8 mg CPP-ACP for 20 minutes 6 times per day for 14 consecutive days;

C. Not chewing gum.

During the treatment period, each subject continued oral hygiene practices consisting of brushing teeth twice daily with fluoride cream toothpaste (supplied) and toothbrush (supplied). Participants chewing gum products were asked to refrain from consuming antimicrobial mints, lozenges, films, and other non-study chewing gums during the treatment period. Similarly, participants who did not chew gum during the treatment period were asked not to consume antimicrobial mints, lozenges, films, and other non-study chewing gums during the treatment period.

Source of participants

Participants were recruited from staff at Melbourne Dental School and Melbourne Dental Clinic at The University of Melbourne. All study participants provided signed informed consent prior to participation.

Participant selection criteria

Inclusion criteria:

To be eligible to participate in this study, subjects met all of the following criteria.

1. Ability to understand informed consent forms, and willingness and ability to read and sign such forms.

2. Age range: 18 to 55 years.

3. Good general health.

4. At least 20 natural teeth.

5. Gum-stimulated total saliva flow rate greater than 1.0 ml/min and unstimulated total saliva flow rate greater than 0.2 ml/min.

6. Willingness to comply with all study procedures and remain responsive for the duration of the study.

Exclusion criteria:

Subjects exhibiting any of the following exclusion criteria at randomization were ineligible for the study:

1. Allergy to milk proteins or other ingredients in gum products.

2. Orthodontic appliances or removable prostheses.

3. More than one incisor with veneers, or prosthetic crowns.

4. Severe oral pathology (including periodontal disease (CPITN ≥ 3) and tumors of soft or hard oral tissues).

5. Chronic diseases with oral findings (e.g., diabetes mellitus [regardless of level of control], human immunodeficiency virus infection or acquired immunodeficiency syndrome, use of medications associated with gingival hyperplasia).

6. Non-healing dental caries.

7. Treatment with antibiotics or anti-inflammatory drugs within 1 month before starting the study.

8. Combination drug therapy with drugs that may interact with the test drug.

9. History of disease requiring antibiotic coverage before invasive dental procedures.

10. Pregnancy/lactation.

test product

Description of test product

Each participant was randomly assigned to one of three treatments:

A. Sugar-free chewing gum containing 56.4 mg CPP-ACP for 20 minutes six times per day for 14 consecutive days;

B. Sugar-free chewing gum containing 18.8 mg CPP-ACP for 20 minutes 6 times daily for 1 consecutive day;

C. Not chewing the gum.

Distribution method

At the beginning of each leg, participants were issued a package containing their assigned treatment for that period, a tube of fluoride cream toothpaste, and a toothbrush. Participants returned any unused gum and all used gum wrappers when they attended the 14-day assessment at the end of each treatment period. The amount of swords distributed and returned was recorded.

How and when to apply

When assigned a gum test product, participants chewed the assigned gum for 20 minutes six times per day for 14 consecutive days at the following times: after breakfast, after morning tea, after lunch, and afternoon tea. ) after, after dinner and before bed.

Randomization and blinding methods

A block randomization schedule was created to ensure that all six treatment combinations (ABC, ACB, BAC, BCA, CAB, and CBA) were equally possible.

The study was investigator-blinded. Throughout the study, clinical investigators and laboratory staff processing plaque samples were blinded to which treatment arm participants were assigned. Because the chewing gums were provided in identical coded packages, neither participants nor study staff knew which chewing gum was assigned. If the participant did not chew the gum during one treatment session, it was not possible to completely blind the participant to treatment assignment. Staff dispensing test materials or supervising their use did not participate in the examination of participants or analysis of plaque samples to minimize potential bias.

A researcher who was not involved in any analysis maintained the master list of code identifications.

Assignment of Participant to Treatment: After baseline testing, participants were assigned a participant number. Participants were randomly assigned to one of three treatments. Randomization was determined from a standard randomization table for number of treatments in parallel studies.

How long processing lasts

Each of the three treatment periods lasted 14 consecutive days. The treatment period was separated by a 14-day washout period.

accompanying procedures

Participants were instructed to continue brushing their teeth twice daily with the provided fluoride cream toothpaste and toothbrush. Participants were required to clean their mouth (except water) during each 14-day treatment period; dental floss; Use of other oral hygiene aids; Patients were instructed to avoid consumption of antimicrobial mints, lozenges, films, and other non-study chewing gums.

Measurements and Observations

efficacy endpoint

clinical examination

At the beginning and end of the treatment period, participants were examined for gingivitis and plaque, and supragingival plaque was collected from the buccal surfaces of the maxillary molars. After completion of examination and supragingival plaque collection, participants underwent supragingival scaling, cleaning, and prophylaxis of all teeth. After completion of the final examination for Leg 3, each participant received a professional fluoride treatment.

Measurement of plaque and gingivitis

Supragingival plaque was assessed using the Turesky modification of the Quigley-Hein Index (Turesky et al 1970 J Periodontol 41(1):41-43). This index is recognized as a reliable estimate of the area of the tooth covered with plaque and is frequently used to evaluate anti-plaque agents. The plaque index was obtained by adding the scores for each tooth and dividing by the number of surfaces examined. Plaque scores associated with unrecovered labial, buccal, and lingual surfaces of all teeth except third molars were assessed. Each surface was assigned a score from 0 to 5.

Gingivitis was measured using the modified gingival index (Lobene et al 1986). This index is a modification of the Roe and Silnis gingival index and allows greater discrimination of mild and moderate gingivitis. In this study, gingival inflammation was scored on a scale of 0 to 4 for gingival tissue associated with four regions (mesial and distal buccal, lingual) of all teeth.

Supragingival plaque collection

All participants had supragingival plaque collected from the buccal surfaces of the maxillary molars (teeth 17, 16, 26, and 27) using sterile scaling instruments. Plaque samples from each tooth were placed in separate sterile tubes, providing four samples per participant.

A one-part label was attached to each container of plaque collected from each participant. The label contained the following information:

· Participant number

· Processing code (Α, Β or C)

· Tooth (17th, 16th, 26th or 27th)

· Processing period (Leg 1, Leg 2 or Leg 3)

· Time (0 or 14 days)

Plaque mass was recorded for each of the four samples from each participant to allow standardization of the DNA extraction procedure (0.2 mg per sample required for DNA extraction). Homogenized plaque samples were stored at -80°C until needed.

Plaque microbiological analysis

Extraction of genomic DNA from dental plaque using a combination of mechanical disruption of cells via a Precellys homogenizer and chemical extraction and purification of genomic DNA via the PowerLyzer PowerSoil DNA Isolation kit (MoBio). did. Quantification of extracted DNA was achieved using the Qubit dsDNA High Sensitivity Assay kit (ThermoFisher), followed by amplification of the V4 variable region of the 16S ribosomal RNA gene and PCR products from each sample. 5 ng DNA was used as a template in the PCR reaction to individually barcode. The barcoded DNA was then sequenced using an Ion Torrent Personal Genome machine and Torrent SuiteTM Software (ThermoFisher).

Analysis of the resulting 16S ribosomal RNA gene sequence allowed identification of the bacterial population present in each sample down to the species level. BAM files created with Torrent SuiteTM software were transferred from Ion Torrent to the local Ion Reporter server, where the premium curated (Ion Reporter Software 16S metagenomics workflow) was used. Premium curated) MicroSEQ ^TM ID 16S rRNA reference database and the curated Greengenes database were used to identify microorganisms present in complex polybacterial samples at the genus or species level. Data were sorted in Microsoft Excel.

statistical methods

Determine Sample Size

Table 1 below shows the mean difference in abundance of Streptococcus sanguinis of 1.5%, 2.0%, 2.5% and 3.0%, standard deviation ranging from 3.5% to 5.5% and estimated sample size for each group for a power of 80%. shows. This assumes a two-tailed paired t-test and α = 0.05. The mean and standard deviation ranges used in sample size calculations were based on a previous pilot study.

[Table 1] Estimated sample size per treatment group

Table 2 shows the size estimates for each group, allowing for a 10% percent attrition rate over the study follow-up period.

[Table 2] Estimated sample size per treatment group

Assuming a standard deviation of 4.5% and a percent reduction of 10%, a sample of 18 participants was recruited such that a mean difference in the abundance of Streptococcus sanguinis of 3.5% would be detected with 80% power.

Statistics General Considerations

Randomization and blinding

Two chewing gums were provided in identical packaging except that each was labeled with a code. Individual pieces of chewing gum had a similar appearance. Code was not released until all statistical analyzes were completed.

After each participant met the participation criteria, IDs were sequentially assigned to the participants.

The study was examiner-blinded. Throughout the study, dental investigators and laboratory staff processing plaque samples were blinded to which treatment arm participants were assigned. Because the chewing gums were provided in identical coded packages, neither participants nor study staff knew which chewing gum was assigned. If the participant did not chew the gum during one treatment session, it was not possible to completely blind the participant to treatment assignment. All participants completed the study.

Compliance criteria: Compliance was determined by reviewing participants' diaries of product use and records of remaining clinical supplies.

Statistical analysis of demographic and baseline characteristics

The primary analysis set was all participants who completed the trial without significant protocol violations.

Descriptive statistics (mean, standard deviation, and range) were calculated for all continuous variables and frequencies for all ordinal variables. All statistical tests were two-sided and a significance level of α = 0.05 was used. All analyzes were performed using the statistical package Stata (StataCorp LP, College Station, TX, USA) statistical software.

Statistical analysis of efficacy data

Individual 16S rDNA sequences generated for each sample were used to classify the microbial lineages present (grouped into species-level taxa) and determine the abundance of each taxon. Differences in bacterial communities over time were determined by measuring changes in relative taxon abundance for each individual.

This enabled comparison of the effect of CPP-ACP on the bacterial composition of plaque. Four samples from each individual allowed determination of within-subject variation in the bacterial composition of plaque.

result

All subjects completed the study and were considered compliant with the protocol. No harmful incidents were reported. Treatment with CPP-ACP did not result in significant changes in the participants' plaque index (Figure 1). CPP-ACP did not affect plaque index, but had a significant effect on bacterial composition, showing a substantial prebiotic effect by promoting the ratio of commensal/beneficial commensals.

In total, over 300 different bacterial taxa were identified from 54 samples. When examining the bacterial composition of supragingival plaque across treatment groups, it was apparent that there were significant changes when comparing CPP-ACP treated legs to untreated legs. 18.8 mg CPP-ACP resulted in a statistically significant increase in the proportion of the following bacterial taxa: Corynebacterium durum (80% increase); Lotia dentocariosa (127% increase); Streptococcus mitis (55% increase) and Streptococcus sanguinis (112% increase) (Table 3). These species are currently considered beneficial commensals because they all possess one or both of the enzyme systems [arginine deiminase and/or nitrate reductase] known to be beneficial to the host. This significant increase in commensal/beneficial species was related to CPP-ACP dose as the increase was significantly higher for the 56.4 mg CPP-ACP dose. The increase in beneficial species was associated with a concomitant decrease in the proportion of pathogenic species (pathogens). This increase in the abundance of beneficial commensals was also reflected in an increase in gingival health, as evidenced by significant CPP-ACP dose-related improvement in gingival indices (Figure 2).

Table 3: Oral species significantly increased by 18.8 mg CPP-ACP are Gram-positive commensals with one or both of the arginine deiminase and nitrate reductase systems that promote homeostasis

[Table 4] Oral species significantly reduced by 18.8 mg CPP-ACP are Gram-negative, inflammatory anaerobes associated with bacterial imbalance.

In conclusion, this randomized controlled clinical study showed that CPP-ACP treatment resulted in a significant increase in the proportion of health-related commensals and improvement in oral health, indicating that CPP-ACP is a prebiotic.

Example 2

SnF ₂ promotes CPP-ACP prebiosis in an ascites microorganism model

Culture of multiple species of oral biofilm

To model supragingival plaque, six representative oral bacterial species: Streptococcus sanguinis (NCTC 7863), Streptococcus mutans Ingbritt, Actinomyces naeslundii (NCTC 10301), and Bailo. Nella parbula (ATCC 17745), Lactobacillus casei (NCDO 161) and Fusobacterium nucleatum (ATCC 108953) (Table 5) were cultured in a constant-depth film fermentor (CDFF; Cardiff University, UK). )) were cultured with ascites microbial biofilms on human enamel substrate. CDFF were housed in a 37°C incubator under anaerobic conditions, which were maintained by a constant flow of 5% CO ₂ in N ₂ at 1 L/h. The CDFF contained 15 removable polytetrafluoroethylene (PTFE) pans on a circular platform rotating at a constant speed of 3 rpm. Biofilms were grown on artificial saliva medium (ASM, 2.5 g/L mucin type II (porcine, stomach, Sigma), 2.0 g/L bacteriological peptone (Oxoid), 2.0 g/L Tryptone (Oxoid), 1.0 g/L yeast extract (Oxoid), 0.35 g/L NaCl, 0.2 g/L KCl, 0.2 mg/L vitamin K, 1 mg/L haemin and 0.1 g/L cysteine hydrochloride) were grown on three enamel blocks (see below) in each PTFE pan. Prior to inoculation, the CDFF pan surface was conditioned with ASM for 24 hours at a flow rate of 10 mL/h (McBain et al (2003) J Appl Microbiol. 94(4):655-66). To mimic in vivo bacterial growth conditions in the oral cavity with dietary sugar intake and to provide a high cariogenic challenge, 30 mL of 1% (w/v) sucrose solution in ASM was administered for 10 minutes four times a day at 4-hour intervals. It was pumped into CDFF at a flow rate of /h.

[Table 5] Bacterial strains used in this study, 16S rRNA gene copy number and species composition of the inoculum.

^a All strains are sourced from the Oral Health Cooperative Research Centre, Melbourne Dental School.

^b 16S rRNA gene copy number was determined by sequence analysis of the genome performed in our laboratory, data not shown.

^c Mean and standard deviation of cell numbers in the inoculum used in this study as determined by qPCR.

Three treatments were applied to the ascites biofilm: 290 ppm fluoride, 2% CPP-ACP and 2% CPP- with 220 ppm fluoride as SnF ₂ and 70 ppm fluoride as NaF (referred to as SnF ₂ ). ACP-SnF ₂ . These treatments were compared to a control where the test solution was replaced with ASM. Each treatment was applied using two 10-minute pulses of test solution in ASM at a flow rate of 30 mL/h. The first pulse began 30 minutes before the first sucrose pulse of the morning, and the second pulse began 3 hours and 30 minutes after the last sucrose pulse of the day.

At days 6, 12, and 19 after inoculation, the four pans were aseptically removed from the CDFF and replaced with blank sterile pans. Enamel blocks from the pan were separated for bacterial counting and transverse microscopy.

Enamel block preparation

Under ethics approval from the University of Melbourne (HREC #1237616), 360 extracted human third molars were obtained and sterilized by exposure to 4.1 kGy of gamma radiation. Enamel blocks were cut from teeth measuring approximately 6 ) and polished using a RotoPol/RotoForce lapping machine. The blocks were placed in a custom CDFF pan to a depth of 100 μm, sealed in place with yellow tacky wax (Kemdent), and sterilized by exposure to 4.1 kGy gamma radiation.

sampling

Floating and loosely attached bacterial cells were first removed from the enamel block by gentle washing with 100 μL of ASM. To harvest biofilm cells on each block, the surface was scraped with a sterile scraper using 1 mL of ASM. Biofilm bacterial cells were sedimented by centrifugation (10,000 g , 10 min), the supernatant was carefully decanted, and the cell pellet was stored at -80°C until required for DNA extraction. The enamel blocks were then used for sectioning and microradiography.

DNA extraction and sequencing of biofilm samples

For each time point, from 8 to 9 enamel blocks using the Powerizer PowerSoil DNA Isolation Kit (MoBio Laboratories) following the manufacturer's “Vacuum Protocol” using a Precellys® bead homogenizer (Bertin Technologies). DNA was extracted separately. Samples were quantified using the QBIT dsDNA HS Assay Kit (Life Technologies) before storage at -80°C.

The Ion Amplicon Library Preparation Fusion Method (Thermo Fisher Scientific) was suitable for amplification of the V4 region of the 16S rRNA gene. PCR consisted of 5 ng of DNA, 1 Performed in a 50 μL reaction volume containing enzyme (Genesearch). The reaction was held at 98°C for 3 minutes to denature the DNA, followed by 17 cycles of amplification at 98°C for 10 seconds, 48°C for 10 seconds, and 72°C for 15 seconds. Forward-primers with barcodes 1 to 62 were designed as specified for the fusion method using GTGCCAGCMGCCGCGGT as the GA spacer and binding region sequence. A single reverse-primer consisting of a reverse adapter, a TC spacer, and a binding domain sequence of GGACTACHVGGGTWTCTAA was used.

Multiplex sequencing of the amplicon library was performed using the Ion Torrent Personal Genome Machine using the Ion OneTouch ^™ 2200 kit, and the Ion PGM ^™ Sequencing 200 Kit v2 chemistry (Thermo Fisher Scientific) extended to 535 cycles.

Sequencing data were analyzed with Ion Torrent software by mapping to known 16S V4 sequences for each of the six species. To adjust for differences in sequencing depth, the total number of reads was normalized by downsampling to the lowest occurring read number.

Confocal laser scanning microscopy

The enamel substrate with attached ascites biofilm was immersed in PBS to rinse the culture medium and non-attached bacterial cells, and then immersed in 4% paraformaldehyde for 30 minutes of fixation at room temperature. Then, it was immersed in PBS to remove paraformaldehyde. In preparation for in situ hybridization, biofilms were embedded in 20% acrylamide with 0.02% ammonium persulfate and 0.8% N,N,N',N' - tetramethylethylenediamine (TEMED) at 4°C. Stored in PBS.

Ascites microbial biofilms were subjected to fluorescence in situ hybridization (FISH) using the following custom species-specific probes. 5' detected Streptococcus mutans probe sequence labeled with Alexa594 ACT CCA GAC TTT CCT GAC, 5' detected AGA CGC AAT CCC CTC CTT labeled with Alexa405 in V. dispar , 5' detected Lacto ACT CTG CCG ACC ATT CTT CT, labeled with Alexa647 in Bacillus casei 5' AGA GAT AGA GTT TCT CTT CGG, 5' labeled with Alexa488 in Streptococcus sanguinis detected 5' in Actinomyces naeslundii CGG TTA TCC AGA AGA AGG GG labeled with Alexa405, and CTA ATG GGA CGC AAA GCT CTC labeled with Alexa647 in 5' detected Fusobacterium nucleatum (Thermofisher, Australia). Biofilms were incubated for 2.5 hours at 46°C with each probe and hybridization buffer containing 10% formamide as described previously (Zainal-Abidin et al (2012). J Proteome Res. 11(9) :4449-4464]). The hybridized biofilm was then immersed in washing buffer (0.45 M NaCl, 20mM Tris-HCl, 5mM EDTA, 0.01% SDS) at 48°C for 25 min and prepared for imaging. Biofilms were visualized on a Zeiss Confocal Laser Scanning Microscope as previously (Zainal-Abidin et al (2012). J Proteome Res. 11(9):4449-4464) and COMSTAT. Parameters for biometric measurements were determined by analysis using software.

scanning electron microscopy

Biofilm samples were prepared for scanning electron microscopy analysis as previously described (Zainal-Abidin et al 2012, supra; Zhu et al (2013) PLoS One. 8(8):e71727).

Transverse microradiography

Transverse microradiography was performed essentially as previously described (Shen et al 2011). The radiographic image of the lesion and the intact enamel next to it are each scanned six times and averaged to provide a demineralized densitometric profile, and the control intact-enamel densitometry profile and volume% mineral (vol% min) content. The profile was calculated. Lesion depth (μm) was determined as the distance to the point where the mineral content reached 95% of the intact enamel value. Integrated mineral loss (IML), expressed in ΔZd, was calculated by trapezoidal integration as the area between the intact-enamel densitometric profile and the demineralized enamel densitometric profile in vol% min.μm.

Biofilm ion analysis

Calcium, phosphorus and tin contents of ascites biofilm samples were determined by inductively coupled mass spectrometry essentially as described previously (Dashper et al 2005).

statistical analysis

Comparison of bacterial species and lesion parameters. The normality of the residuals was checked using a QQ plot, and the Shapiro-Wilk test for normality and homogeneity of variance was tested using Levene's test. Because the residuals for bacterial species composition did not approach a normal distribution even after Box-Cox transformation using Minitab version 17 software (Minitab Inc., State College, PA, USA), differences in species proportions were analyzed using the Kruskal-Wallis method. -Wallis) test was used to analyze more than two samples. Differences between treatments were determined using a post hoc Wilcoxon Rank Sum test with Bonferroni correction (Sokal and Rohlf (1969). Biometry San Francisco: WH Freeman and Co. ]). For differences in lesion parameters, LD at day 6 was determined on Box-Cox transformed data using one-way analysis of variance, and pairwise differences were determined using post hoc Tukey tests. Differences between ΔZd values on day 6 and both LD and ΔZd values on days 12 and 19 were determined using the Kruskal-Wallis test and post hoc Wilcoxon rank sum test with Bonferroni correction. All statistical tests were performed using SPPS version 22 software (IBM SPSS Inc., IL, USA). In all cases, α was set to 0.05.

result

Development of ascites microbial biofilm model

Many of all six bacterial species were present in the ascites inoculum as determined by 16s rRNA gene analysis (Table 5). Ascites biofilms were rapidly established on intact human enamel substrates in constant-depth film fermentors (CDFFs) after inoculation when grown in the presence of artificial saliva medium (ASM) with frequent exposure to sucrose. Although Fusobacterium nucleatum was not present in excess of 0.01% of the total bacteria present, all six bacterial species were detectable in the ascites biofilm of all four biological replicates of the control group at all time points. Actinomyces naeslundii increased over time from less than 2% on day 6 to over 18% of total bacteria on day 19 (Figure 3). The proportion of Lactobacillus casei increased rapidly over the course of the experiment and was present at an average of 16% of the total bacterial population by day 19. The proportion of Streptococcus sanguinis decreased from 36% of total biofilm bacteria at day 6 to less than 11% by day 19. There was no clear trend in the proportion of Streptococcus mutans over time, and it is likely that this species remained relatively stable. Streptococcus mutans was the most abundant species in the biofilm, accounting for 48 to 64% of the total bacterial population (Figure 3).

Demineralization of the initially intact human enamel matrix was produced by exposure of enamel blocks to sucrose-pulsed ascitic biofilms. Similar enamel lesions were created that maintained an intact surface layer and were associated with the initial carious lesions seen in vivo (Figure 4). The ascitic biofilm also had a structure similar to supragingival plaque when imaged by scanning electron microscopy (Figure 4). Lesion formation proceeded in a linear fashion after an initial lag phase with an average constant rate of mineral loss of 225.1 vol% min.μm/day from day 6 to day 19 (Figure 4). The average lesion depth at day 19 was 87.1 ± 8.4 μm and the average integrated mineral loss (ΔZd) was 3575.6 ± 562.0 vol% min.μm (Table 6).

[Table 6] SnF for integrated mineral loss (ΔZd) and lesion depth (LD) of subsurface lesions formed in the enamel substrate of the ascites biofilm caries model. ₂ , CPP-ACP and CPP-ACP/SnF ₂ influence.

Day 6: Same superscripts in the LD column indicate significant differences; ( ^ac p <0.05; ^b p < 0.001): Identical superscripts in the ΔZd column indicate significant differences ( ^a p < 0.05). All other differences are not significant (p > 0.05).

Day 12: Same superscripts within columns indicate significant differences. LD - All differences are significantly different (p < 0.05). ΔZd - All differences are significantly different (p < 0.05) except between CPP-ACP and CPP-ACP+SnF ₂ (p > 0.05).

Day 19: Same superscripts in columns indicate significant differences - LD and ΔZd - all differences are significantly different (p < 0.05) except between SnF ₂ and CPP-ACP (p > 0.05).

SnF on the composition of ascites microbial biofilms ₂ and the impact of CPP-ACP

*Treatment of ascites microbial biofilm twice a day with SnF ₂ significantly reduced the abundance of Actinomyces naeslundii and increased the abundance of Streptococcus sanguinis compared to the control group on day 19 (Figure 5 , Table 7). Treatment of ascitic biofilms with CPP-ACP twice daily resulted in more significant changes in the composition of ascitic biofilms, especially up to 19 days after inoculation. The proportions of Streptococcus mutans and Veillonella parbula remained relatively constant, but Lactobacillus casei decreased sharply, and both Streptococcus sanguinis and Fusobacterium nucleatum increased significantly compared to the control group. (Figure 5). The largest change in relative abundance was a consistent >95% decrease in Lactobacillus casei, while Fusobacterium nucleatum showed the largest average increase of 355% at day 19 (Figure 5b, Table 7). The combination of CPP-ACP and SnF ₂ had a more significant effect on the bacterial composition of the ascites microbial biofilm (Figure 5 and Table 7). There was a notable decline in the acid-producing and acid-tolerant species Streptococcus mutans, Actinomyces naeslundii and Lactobacillus casei as well as Veillonella parbula. The acid-sensitive species Fusobacterium nucleatum increased rapidly to 34% of the total bacteria, becoming the second most abundant species in the ascites biofilm, and Streptococcus sanguinis also increased in abundance by 50%. All six bacterial species were always detectable in the treated biofilm. Confocal laser scanning microscopy of CPP-ACP/SnF ₂ treated ascites biofilms using specific dyes for four bacterial species confirmed the emergence of Fusobacterium nucleatum as a major component of the community (Figure 5).

Table 7: Impact of the four treatments on bacterial species composition as a percentage of total bacteria in the biofilm.

*Mean ± standard deviation. Proportion of bacterial species in ascites biofilms exposed to four treatments at day 19.

Identical superscripts in the ^abcde column indicate significant differences (p < 0.05). n = 5 to 21.

All comparisons across treatments were performed with the Kruskal-Wallis test (see overall p-values) and pairwise differences between treatments were determined using the post hoc Wilcoxon rank sum test with Bonferroni correction.

SnF for enamel demineralization ₂ and the impact of CPP-ACP formulations.

Treatment of the ascites biofilm with SnF ₂ resulted in a significant 50.2% decrease in the demineralization rate to 112.1 vol% min.㎛/day between days 6 and 19 (Table 6). This reduction was not statistically different from that seen with the CPP-ACP treatment, which also resulted in a 50.2% reduction in demineralization rate (112.1 vol% min.μm/day) over the same period. However, the reduction in demineralization rate to 64.1 vol% min.μm/day was significantly higher (72%) for the combined SnF ₂ /CPP-ACP treatment (Table 6). ICP-MS analysis of the biofilm showed a four-fold increase in calcium and a three-fold increase in phosphorus when CPP-ACP was used with SnF ₂ . Interestingly, SnF ₂ treatment resulted in a two-fold increase in both calcium and phosphorus compared to the control. The stannous concentration peaked at 1.0 nmol/mg biofilm wet weight during both SnF ₂ formulation and CPP-ACP-SnF ₂ treatment. However, this concentration was reached earlier with CPP-ACP-SnF ₂ treatment.

discussion

The six bacterial biofilm communities generated in CDFF by four pulses of sucrose per day were dominated by more acid-producing and acid-resistant species, and thus, cariogenic species Streptococcus mutans and Actinomyces na were present at day 19. Eshlundii and Lactobacillus casei together comprised 85% of the ascites microbial biofilm. This percentage was an increase from 55% on day 6. When examining only the ratio of Actinomyces naeslundii and Lactobacillus casei in the control biofilm, there was an increase from 2.5% to 34% of total bacterial cells from day 6 to day 19 (Figure 3). This represents a highly acidic and cariogenic environment, consistent with the low levels of the neutrophil species Fusobacterium nucleatum and the rapid, consistent and reproducible demineralization rates in this model (Figure 4).

After establishing the reproducibility of the model, the effect of twice daily addition of 290 ppm fluoride as a mixture of SnF ₂ (220 ppm F):NaF (70 ppm F) was studied, which was compared to the current method using SnF ₂ /NaF. It was chosen to represent a 5-fold dilution in saliva of 1450 ppm F toothpaste. In the study, Actinomyces naeslundii significantly decreased and Streptococcus sanguinis significantly increased in relative abundance in the ascites biofilm during SnF ₂ treatment. Sn accumulated in the ascites biofilm to a concentration of 119 ppm. SnF ₂ reduced enamel demineralization rates by 50% over 19 days of twice-daily exposure in this study, and the main mechanism appears to be related to the action of F ion-promoted remineralization.

Addition of CPP-ACP not only resulted in a significant 50% reduction in demineralization rate, but also inhibited the emergence of highly acidogenic Lactobacillus casei. Additionally, there was a reproducible increase in the abundance of the acid-sensitive, beneficial commensals Fusobacterium nucleatum and Streptococcus sanguinis (Figure 5, Table 7). This indicates that CPP-ACP had a prebiotic effect on biofilm development in this model.

The enhanced prebiotic effect on the bacterial composition of ascites biofilms by CPP-ACP and SnF ₂ was noted with respect to all three acid-producing and acid-tolerant species with decreasing abundances. Veillonella parbula, which may indicate a decrease in lactate due to inhibition of glycolysis, also decreased, and Fusobacterium nucleatum became a major component of the biofilm. These changes were associated with a highly significant 72% inhibition of enamel demineralization rates, which would translate into significant improvements in oral health. The additive effect of SnF ₂ and CPP-ACP in promoting prebiosis, as demonstrated in Example 3, crosslinks CPP-ACP and further promotes prebiotics to ascitic biofilms and intraoral/tooth surfaces. This was due to the ability of tin ions (Sn ²⁺ ) to be transmitted well.

It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

Claims (36)

Increasing commensal oral bacteria containing phosphopeptide stabilized amorphous calcium phosphate (PP-ACP) or phosphopeptide stabilized amorphous calcium phosphate (PP-ACP) A composition intended for use. According to paragraph 1,
The composition, wherein the commensal oral bacteria are one or more species expressing arginine deiminase or nitrate reductase. According to paragraph 2,
The commensal oral bacteria include Corynebacterium durum, Rothia dentocariosa , Streptococcus mitis, Streptococcus sanguinis , and Fusobacterium nu. A composition that is one or more of Fusobacterium nucleatum . According to paragraph 1,
The composition further comprises reducing pathogenic oral bacteria. According to paragraph 4,
The composition, wherein the pathogenic oral bacteria are one or more associated with gingival inflammation, gingivitis, chronic gingivitis, or periodontal disease. According to clause 5,
A composition wherein the pathogenic oral bacteria are acid-producing, tolerant, or inflammatory. According to clause 6,
A composition wherein the pathogenic oral bacteria are inflammatory. According to clause 6,
The pathogenic oral bacteria include Streptococcus mutans, Actinomyces naeslundii , Veillonella parvula, Lactobacillus casei, and Porphyromonas ginseng. Porphyromonas gingivalis , Tannerella forsythia, Treponema denticola, Leptotrichia wadei , Leptothrichia shahii , Leptothrichia A composition, one or more selected from Leptotrichia buccalis and Lautropia mirabilis . The composition according to claim 1, wherein the use is further to inhibit oral dysbiosis. According to paragraph 1,
The composition of claim 1, wherein the subject has no detectable dental surface or subsurface lesions. According to paragraph 1,
A composition, wherein the subject has undergone a dental procedure. According to clause 11,
The composition of claim 1, wherein the dental procedure is selected from debridement, scaling, and root planning. According to any one of claims 1 to 12,
The phosphopeptide stabilized amorphous calcium phosphate (PP-ACP) or phosphopeptide stabilized amorphous calcium fluoride phosphate (PP-ACFP) is casein phosphopeptide stabilized amorphous calcium phosphate (casein PP-ACP, CPP-ACP). or casein phosphopeptide stabilized amorphous calcium fluoride phosphate (casein PP-ACFP, CPP-ACFP). According to any one of claims 1 to 12,
A composition wherein the PP-ACP or PP-ACFP is formulated to be administered to an individual for 5 to 60 minutes. According to any one of claims 1 to 12,
A composition wherein the PP-ACP or PP-ACFP is formulated to be administered 4 to 6 times a day. According to any one of claims 1 to 12,
A composition wherein the PP-ACP or PP-ACFP is formulated to be administered over a period of 1 to 2 weeks. According to any one of claims 1 to 12,
The PP-ACP or PP-ACFP is used in cream-type toothpaste, tooth powder and liquid toothpaste, mouthwash, mouthwash, oral spray, varnish, dental cement, troches, chewing gum, dental A composition formulated as any one of a paste for use, a cream for gingival massage, a tablet for gargling, and a dairy product. According to clause 17,
A composition wherein the PP-ACP or PP-ACFP is formulated as chewing gum. According to clause 18,
The composition of claim 1, wherein the chewing gum contains at least 15 mg to 60 mg of PP-ACP or PP-ACFP. According to clause 19,
The composition of claim 1, wherein the chewing gum contains 18.8 mg or 56.4 mg of PP-ACP or PP-ACFP. Pharmaceuticals for reducing gingival inflammation, comprising phosphopeptide stabilized amorphous calcium phosphate (PP-ACP) or phosphopeptide stabilized amorphous calcium phosphate (PP-ACP) enemy composition. According to clause 21,
A composition for reducing gingival inflammation, wherein the subject has mild, moderate, or severe inflammation. According to clause 21,
A composition for reducing gingival inflammation, wherein the subject has a Modified Gingival Index score of 1 to 4. Pharmaceutical composition for treating gingivitis comprising phosphopeptide stabilized amorphous calcium phosphate (PP-ACP) or phosphopeptide stabilized amorphous calcium phosphate (PP-ACP) . Pharmaceuticals for the treatment of chronic gingivitis containing phosphopeptide stabilized amorphous calcium phosphate (PP-ACP) or phosphopeptide stabilized amorphous calcium phosphate (PP-ACP) Composition. Pharmaceutical composition for treating periodontitis comprising phosphopeptide stabilized amorphous calcium phosphate (PP-ACP) or phosphopeptide stabilized amorphous calcium phosphate (PP-ACP) . The method according to any one of claims 21 and 24 to 26,
A pharmaceutical composition, wherein the subject is an individual who has undergone a dental procedure. According to clause 27,
The pharmaceutical composition of claim 1, wherein the dental procedure is selected from debridement, scaling, and root planning. The method according to any one of claims 21 and 24 to 26,
The phosphopeptide stabilized amorphous calcium phosphate (PP-ACP) or phosphopeptide stabilized amorphous calcium fluoride phosphate (PP-ACFP) is casein phosphopeptide stabilized amorphous calcium phosphate (casein PP-ACP, CPP-ACP). or casein phosphopeptide stabilized amorphous calcium fluoride phosphate (casein PP-ACFP, CPP-ACFP). The method according to any one of claims 21 and 24 to 26,
A pharmaceutical composition wherein the PP-ACP or PP-ACFP is formulated to be administered to an individual for 5 to 60 minutes. The method according to any one of claims 21 and 24 to 26,
A composition wherein the PP-ACP or PP-ACFP is formulated to be administered 4 to 6 times a day. The method according to any one of claims 21 and 24 to 26,
A composition wherein the PP-ACP or PP-ACFP is formulated to be administered over a period of 1 to 2 weeks. The method according to any one of claims 21 and 24 to 26,
The PP-ACP or PP-ACFP is used in cream-type toothpaste, tooth powder and liquid toothpaste, mouthwash, mouthwash, oral spray, varnish, dental cement, troches, chewing gum, dental A composition formulated as any one of a paste for use, a cream for gingival massage, a tablet for gargling, and a dairy product. According to clause 33,
A composition wherein the PP-ACP or PP-ACFP is formulated as chewing gum. According to clause 34,
The composition of claim 1, wherein the chewing gum contains at least 15 mg to 60 mg of PP-ACP or PP-ACFP. According to clause 34,
The composition of claim 1, wherein the chewing gum contains 18.8 mg or 56.4 mg of PP-ACP or PP-ACFP.